Car peptide for homing, diagnosis and targeted therapy for pulmonary and fibrotic disorders

ABSTRACT

Disclosed are compositions and methods useful for delivering targeted therapies for pulmonary diseases, fibrotic disorders and cancer. The compositions and methods are based on peptide sequences that selectively bind to and home to diseased tissue and enable targeted therapies to effect a beneficial therapeutic result. The disclosed targeting is useful for delivering therapeutic and detectable agents to diseased tissue in an animal.

CROSS REFERENCES

This application is a Divisional application claiming the benefit ofpriority from U.S. National Stage application Ser. No. 13/581,457 filedFeb. 26, 2013, now U.S. Pat. No. 9,180,161, which claims priority under35 U.S.C. 371 from International Patent Application No. PCT/US2011/26535filed Feb. 28, 2011, which claims the benefit of priority from U.S.Provisional Patent Application No. 61/308,826, filed Feb. 26, 2010, theentire contents of which are herein incorporated by reference.

GOVERNMENT SUPPORT STATEMENT

This invention was made with government support under grant 1 R41HL088771 from the National Heart Lung Blood Institute of the NIH. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of targetedtherapies, more specifically, to therapies that selectively targetdiseased pulmonary and fibrotic tissue.

BACKGROUND OF THE INVENTION

Tissue regeneration, inflammation and tumors induce the growth of newblood vessels from pre-existing ones. This process, angiogenesis, is avital requirement for wound healing as the formation of new bloodvessels allows a variety of mediators, nutrients, and oxygen to reachthe healing tissue (Martin 1997, Singer & Clark 1999, Falanga 2006,Folkman 2006). Newly formed blood vessels differ in structure frompre-existing vasculature. Such differences have been extensivelycharacterized by comparing tumor vasculature to normal vessels(Ruoslahti, 2002). Angiogenic vessels in non-malignant tissues and inpre-malignant lesions share markers with tumor vessels (Gerlag et al,2001), but distinct markers also exist (Hoffman et al., 2003; Joyce etal., 2003).

Regarding tissue injuries, substantive basic science and clinicalresearch have been conducted to evaluate the mechanisms of woundhealing, the efficacy of various modalities for treatment of wounds, andthe best methods for diagnosing wound infection. Tissue injuries causedby trauma, medical procedures, and inflammation are a major medicalproblem. Systemic medication is available for most major medicalconditions, but therapeutic options in promoting tissue regeneration arelargely limited to local intervention. As deep injuries and multiplesites of injury often limit the usefulness of local treatment, systemicapproaches to tissue regeneration are valuable.

A major problem limiting tissue regeneration is scar formation. Theresponse to tissue injury in adult mammals seems to be mainly focused onquick sealing on the injury. Fibroblast (astrocyte, smooth muscle cell)proliferation and enhanced extracellular matrix production are the mainelement of the scar formation, and the scar prevents tissueregeneration. In contrast, fetal tissues heal by a process that restoresthe original tissue architecture with no scarring. Transforming growthfactor.beta. (TGF-.beta.) is a major factor responsible for impairedtissue regeneration, scar formation and fibrosis (Werner and Grose 2002;Brunner and Blakytny 2004; Leask and Abraham 2004).

A major hurdle to advances in treating cancer is the relative lack ofagents that can selectively target the cancer while sparing normaltissue. For example, radiation therapy and surgery, which generally arelocalized treatments, can cause substantial damage to normal tissue inthe treatment field, resulting in scarring and loss of normal tissue.Chemotherapy, in comparison, which generally is administeredsystemically, can cause substantial damage to organs such as the bonemarrow, mucosae, skin and small intestine, which undergo rapid cellturnover and continuous cell division. As a result, undesirable sideeffects such as nausea, loss of hair and drop in blood cell count oftenoccur when a cancer patient is treated intravenously with achemotherapeutic drug. Such undesirable side effects can limit theamount of a drug that can be safely administered, thereby hamperingsurvival rate and impacting the quality of patient life. For decades,researchers have examined avenues to increase targeted specificity oftherapeutics against only the disease, thereby preserving normalcellular integrity.

One manner by which therapeutic specificity may be increased is bytargeting diseases at the cellular level. More specifically,therapeutics may be enhanced by interacting directly with thosecomponents at the level of the cell surface or membrane. Thesecomponents include, among others, laminin, collagen, fibronectin andother proteoglycans. Proteoglycans are proteins classified by aposttranslational attachment of polysaccharide glycosaminoglycan (GAG)moieties each comprised of repeating disaccharide units. Onemonosaccharide of the disaccharide repeat is an amino sugar withD-glucosamine or galactosamine, and the other unit is typically, but notalways, a uronic acid residue of either D-glucuronic acid or iduronicacid. Both units are variably N- and O-sulfated, which adds to theheterogeneity of these complex macromolecules. They can be foundassociated with both the extracellular matrix and plasma membranes. Themost common GAG structures are dermatan sulfate (DS), chondroitinsulfate (CS), heparan sulfate (HS), keratan sulfate (KS), hyaluronicacid (HA), and heparin; representative structures for each disaccharideare shown below.

These unbranched sulfated GAGs are defined by the repeating disaccharideunits that comprise their chains, by their specific sites of sulfation,and by their susceptibility to bacterial enzymes known to cleavedistinct GAG linkages. All have various degrees of sulfation whichresult in a high density of negative charge. Proteoglycans can bemodified by more than one type of GAG and have a diversity of functions,including roles in cellular adhesion, differentiation, and growth. Inaddition, cell surface proteoglycans are known to act as cellularreceptors for some bacteria and several animal viruses, including;foot-and-mouth disease type O virus, HSV types 1 and 2 and dengue virus.Accordingly, it would be advantageous from a therapeutic perspective todesign agents which may be used at the cell surface level.

A major function of cell surface proteoglycans is in cell adhesion andmigration, dynamic processes that are mediated through interactionsbetween the proteoglycan GAG chains and extracellular matrix (ECM)components, such as laminin, collagen, and fibronectin. Proteoglycansalso occur as integral components of basement membranes in mostmammalian tissues. Interactions of these macromolecules with other ECMconstituents contribute to the general architecture and permeabilityproperties of the basement membrane, and thus these GAGs play astructural role. Proteoglycans and GAGs play a critical role in thepathophysiology of basement membrane-related diseases, includingdiabetes, atherosclerosis, and metastasis. In addition, cell-specificgrowth factors and enzymes are immobilized in the ECM and at the cellsurface are bound to GAGs. As such, GAGs localize proteins and enzymesat their site of action to facilitate their physiological functions andin some cases prevent their proteolytic degradation. Proteoglycans andGAGs have been shown to regulate protein secretion and gene expressionin certain tissues by mechanisms involving both membrane and nuclearevents, including the binding of GAGs to transcription factors (Jackson,R. L. 1991). Limited information is available on the factors thatregulate the expression of proteoglycans and their associated GAGs.There is a need in the art to develop cell-penetrating agents which bindto cell surface proteoglycans in order to have disease-specificefficacy.

US Patent Application Publication No. 20090036349 discloses a novelcomposition that selectively binds to regenerating tissue, wound sitesand tumors in animals. In vivo screening of phage-displayed peptidelibraries was used to probe vascular specialization. This screeningmethod resulted in the identification of several peptides thatselectively target phage to skin and tendon wounds. One peptide inparticular was identified and contains the following sequence: CARSKNKDC(CAR) (SEQ ID NO:1). CAR displays homology to heparin-binding sites invarious proteins, and binds to cell surface heparan sulfate and heparin.More specifically, CAR binds to glycosaminoglycan moieties in cellsurface heparan sulfate proteoglycans (HSPGs) (Jarvinen and Ruoslahti2007), and other cell-penetrating peptides have also mediated theirentry into cells through binding to HSPGs (Poon and Gariépy 2007). HSPGsfine-tune mammalian physiology and orchestrate metabolism, transport,information transfer, support and regulation at the systemic level, aswell as the cellular level (Bishop, Schuksz and Esko 2007).Overexpression of HSPG biosynthetic enzymes result in distinct heparansulfate sulfation patterns (Pikas, Erikson and Kjellen 2000). Theoverexpression of HSPG biosynthetic enzymes have not been previouslydetected in a disease in which the co-administration of a cellpenetrating peptide along with a bioactive agent which results in thedisease-selective action of the co-administration of the peptide/agentcombination.

There is a need for selectively targeting specific sites of actionthrough co-administration of a targeting peptide with a bioactive agentin order to increase the efficacy of the combination therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of CAR-FITC staining between control ratpulmonary cross-sections and monocrotaline (MCT) induced hypertensivepulmonary artery.

FIG. 2 shows a hematoxylin and eosin (H&E) stain of rat pulmonary arteryillustrating the vascular remodeling and thicker arterial walls seen inthe MCT induced hypertensive pulmonary artery as compared to control ratpulmonary artery.

FIG. 3 shows a H&E stain of rat pulmonary aveoli and arterioles showingmore remodeling and alveolar macrophages in MCT rat as compared tocontrol.

FIG. 4 shows alveolar macrophages take up the stain for CAR-FITC in theMCT rat.

FIGS. 5-13 depict varying distributions of CAR and the control peptideCARM in tissues from the late stage SU5146/hypoxia/normoxia exposedrats. Tissue samples were collected 2 hours after injections ofFAM-labeled CAR or CARM peptide via tail vein (1 mg/300 g body weight).Low power magnification of lung tissues (FIG. 5), high powermagnification of severely remodeled (Grade 4) pulmonary arterial lesions(FIGS. 6-10), low power and high power magnification photos of liver(FIG. 11), spleen (FIG. 12), kidney (FIG. 13).

FIG. 14 shows a distribution of CAR and VCAM1 staining in mouse lungs 3days after bleomycin injection in acute lung injury model. Both CAR andVCAM1 stain the interstitium and inflammatory cells although CARstaining appears to be more intense.

FIG. 15 shows a distribution of CAR staining 14 days after bleomycininjection in pulmonary fibrosis model. Interstitial and inflammatorystaining is seen in areas of lung fibrosis.

FIG. 16 shows a distribution of CAR staining 14 days after bleomycininjection in areas of dense fibrosis and spared areas showing littlestaining.

FIG. 17 shows a distribution of VCAM1 staining 14 days after bleomycininjection in areas of mild fibrosis and spared areas showing littlestaining.

FIG. 18 shows a blood pressure tracing of catheterizedSU5416/hypoxia/normoxia-exposed rats with PAH. Acute iv effects offasudil (10 mg/kg) alone (A and B) and of CAR (1 mg/300 g rat)+fasudil(1 mg/kg) mixture (C and D) on right ventricular (RVSP, A and C) andleft ventricular systolic pressure (LVSP, B and D).

FIG. 19 shows the effects of acute administration of fasudil with (+CAR)and without co-administration of CAR (1 mg/300 g rat) (−CAR) on right(RVSP) and left ventricle systolic pressure (LVSP). Vasodilator effectswere expressed as % reduction of baseline pressure. Values are means ofn=1-2 each.

FIG. 20 shows a continuous infusion of CAR+fasudil and the effects onthe RVSP and LVSP.

FIG. 21 shows the pulmonary selective enhancement of fasudil whenco-administered with CAR in the severe occlusive PAH rat model. Fasudildosing ranges from 1 to 10 mg/kg. RVSP and SAP were analyzed afterfasudil dosing, both with and without CAR.

FIG. 22 shows a blood tracing experiment with fasudil co-administeredwith CARK. Fasudil was dosed at 1 mg/kg and CARK dosed at 3 mg/kg.Pressure measurements were observed at RVSP (mmHg) and SAP (mmHg).

FIG. 23 shows blood tracing experiments with fasudil co-administeredwith CARK. Fasudil was dosed at 1 mg/kg and CARK dosed at 3 mg/kg.Pressure measurements were observed at RVSP (mmHg) and LVSP (mmHg).

FIG. 24 shows a blood tracing experiment with fasudil administered aftercessation of CARK infusion. Fasudil was dosed at 10 mg/kg. Pressuremeasurements were observed at RVSP (mmHg) and LVSP (mmHg).

FIG. 25 shows a blood tracing experiment with imatinib co-administeredwith CAR. Imatinib was dosed at 10 mg/kg and CAR dosed at 3 mg/kg.

FIG. 26 shows gene expression levels of HS2ST1 (heparan sulfate2-O-sulfotransferase 1) in the PAH model. HS2ST1 catalyzes the transferof sulfate to the C2-position of selected hexuronic acid residues withinthe maturing heparan sulfate.

FIG. 27 shows gene expression levels of EXT1 (exostosin 1) in the PAHmodel. EXT1 is an endoplasmic reticulum-resident type II transmembraneglycosyltransferase involved in the chain elongation step of heparansulfate biosynthesis.

FIG. 28 shows gene expression levels of GLT8D2 (glycosyltransferase 8domain containing 2) in the PAH model. GLT8D2 is an enzyme involved inHSPG biosynthesis.

FIG. 29 shows gene expression levels of NDST1 (Heparan sulfateN-deacetylase/N-sulfotransferase) in the PAH model. NDST1 is a HSPGbiosynthetic enzyme.

FIG. 30 shows gene expression levels of OGT (O-linkedN-acetylglucosamine (O-GlcNAc) transferase) in the PAH model. OGTcatalyzes the addition of a single N-acetylglucosamine in O-glycosidiclinkage to serine or threonine residues of intracellular proteinsincluding HSPGs.

FIG. 31 shows the molecular structure of CARK (in upper left) andsubstitutional variants of CARK in which each of the other 19 aminoacids are substituted for the N terminus cysteine (C).

FIG. 32 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant AARSKNK (SEQ ID NO: 3) (left images) in which alanine (A) hasbeen substituted for the N terminus cysteine (C).

FIG. 33 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant SARSKNK (SEQ ID NO: 5) (left images) in which serine (S) hasbeen substituted for the N terminus cysteine (C).

FIG. 34 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant MARSKNK (SEQ ID NO: 7) (left images) in which methionine (M) hasbeen substituted for the N terminus cysteine (C).

FIG. 35 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant VARSKNK (SEQ ID NO: 9) (left images) in which valine (V) hasbeen substituted for the N terminus cysteine (C).

FIG. 36 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant HARSKNK (SEQ ID NO: 12) (left images) in which alanine (A) hasbeen substituted for the N terminus cysteine (C).

FIG. 37 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant TARSKNK (SEQ ID NO: 6) (left images) in which threonine (T) hasbeen substituted for the N terminus cysteine (C).

FIG. 38 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant NARSKNK (SEQ ID NO: 4) (left images) in which asparagine (N) hasbeen substituted for the N terminus cysteine (C).

FIG. 39 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant GARSKNK (SEQ ID NO: 8) (left images) in which glutamic acid (G)has been substituted for the N terminus cysteine (C).

FIG. 40 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant LARSKNK (SEQ ID NO: 10) (left images) in which leucine (L) hasbeen substituted for the N terminus cysteine (C).

FIG. 41 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant IARSKNK (SEQ ID NO: 11) (left images) in which isoleucine (I)has been substituted for the N terminus cysteine (C).

FIG. 42 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant QARSKNK (SEQ ID NO: 16) (left images) in which glutamine (Q) hasbeen substituted for the N terminus cysteine (C).

FIG. 43 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant FARSKNK (SEQ ID NO: 13) (left images) in which alanine (A) hasbeen substituted for the N terminus cysteine (C).

FIG. 44 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant EARSKNK (SEQ ID NO: 17) (left images) in which phenylalanine (F)has been substituted for the N terminus cysteine (C).

FIG. 45 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant WARSKNK (SEQ ID NO: 14) (left images) in which tryptophan (W)has been substituted for the N terminus cysteine (C).

FIG. 46 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant YARSKNK (SEQ ID NO: 15) (left images) in which tyrosine (Y) hasbeen substituted for the N terminus cysteine (C).

FIG. 47 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant DARSKNK (SEQ ID NO: 18) (left images) in which aspartic acid (D)has been substituted for the N terminus cysteine (C).

FIG. 48 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant PARSKNK (SEQ ID NO: 21) (left images) in which proline (P) hasbeen substituted for the N terminus cysteine (C).

FIG. 49 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant KARSKNK (SEQ ID NO: 20) (left images) in which lysine (K) hasbeen substituted for the N terminus cysteine (C).

FIG. 50 shows a conformational comparison between the electrostaticpotential surface structure of CARK (right images) and a substitutionalvariant RARSKNK (SEQ ID NO: 19) (left images) in which arginine (R) hasbeen substituted for the N terminus cysteine (C).

FIG. 51 shows the molecular structure in sticks of a CARK 8-merdendrimer composed of 8 copies of CARK connected to a polyamidoaminecore.

FIG. 52 shows the molecular structure in space filling balls of a CARK8-mer dendrimer composed of 8 copies of CARK connected to apolyamidoamine core.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides for a compositioncomprising: (a) a targeting peptide comprising at least one amino acidsequence having substantial identity to a sequence selected from thegroup consisting of SEQ ID NO:1 and SEQ ID NO:2, and (b) at least onebioactive agent which conveys a measurable therapeutic benefit to adisease. Preferably, the disease is selected from the group consistingof pulmonary hypertension, interstitial lung disease, acute lung injury(ALI), acute respiratory distress syndrome (ARDS), sepsis, septic shock,sarcoidosis of the lung, pulmonary manifestations of connective tissuediseases, including systemic lupus erythematosus, rheumatoid arthritis,scleroderma, and polymyositis, dermatomyositis, bronchiectasis,asbestosis, berylliosis, silicosis, Histiocytosis X, pneumotitis,smoker's lung, bronchiolitis obliterans, the prevention of lung scarringdue to tuberculosis and pulmonary fibrosis, other fibrotic diseases suchas myocardial infarction, endomyocardial fibrosis, mediastinal fibrosis,myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis,pneumoconiosis, nephrogenic systemic fibrosis, keloid, arthrofibrosis,adhesive capsulitis, radiation fibrosis, fibrocystic breast condition,liver cirrhosis, hepatitis, liver fibrosis, nonalcoholic fatty liverdisease, nonalcoholic steatohepatitis, sarcoidosis of the lymph nodes,or other organs, inflammatory bowel disease, crohn's disease, ulcerativecolitis, primary biliary cirrhosis, pancreatitis, interstitial cystitis,chronic obstructive pulmonary disease, pneumoconiosis, autoimmunediseases, angiogenic diseases, wound healing, infections, traumainjuries and systemic connective tissue diseases including systemiclupus erythematosus, rheumatoid arthritis, scleroderma, polymyositis,and dermatomyositis.

Alternatively, the targeting peptide comprises the amino acid sequenceof SEQ ID NO:1. In another embodiment, the targeting peptide comprisesthe amino acid sequence of SEQ ID NO:2. In yet another embodiment, thetargeting peptide is animal, bacterial, viral or synthetic in origin. Analternative embodiment is disclosed, wherein the targeting peptide andthe at least one bioactive agent are bound together. Alternatively, thetargeting peptide and the at least one bioactive agent are in an unboundconfiguration. In yet another embodiment, the at least one bioactiveagent is specific for the disease to be treated. Alternatively, the atleast one bioactive agent is non-specific for the disease to be treated.

In another embodiment, the present invention describes a method ofpreventing or treating a disease comprising the steps of: (a) providinga targeting peptide comprising at least one amino acid sequence havingsubstantial identity to a sequence selected from the group consisting ofSEQ ID NO:1 and SEQ ID NO:2; (b) providing at least one bioactive agentwhich conveys a measurable therapeutic benefit to the disease; (c)co-administering a composition comprising (a) and (b) to an animal inneed thereof; and (d) measuring the therapeutic benefit to the animal.Alternatively, the disease to be treated is pulmonary or fibrotic. Inyet another alternative embodiment, the disease to be treated is cancer.In yet another embodiment, the disease to be treated is an autoimmunedisease. In yet a further embodiment, the disease to be treated is aninflammatory disease. Alternatively, the targeting peptide of thedisclosed method comprises on the amino acid sequence of SEQ ID NO:1. Inanother embodiment, the targeting peptide of the disclosed methodcomprises on the amino acid sequence of SEQ ID NO:2. In yet anotherembodiment, the targeting peptide is animal, bacterial, viral orsynthetic in origin. An alternative embodiment is disclosed, wherein thetargeting peptide and the at least one bioactive agent are in boundtogether. Alternatively, the targeting peptide and the at least onebioactive agent are in an unbound configuration. In yet anotherembodiment, the at least one bioactive agent is specific for the diseaseto be treated. Alternatively, the at least one bioactive agent isnon-specific for the disease to be treated.

In still another embodiment, the present invention describes a method ofdetecting an enhanced targeting effect of cell penetrating peptides inan animal comprising the steps of: (a) analyzing a sample from theanimal for an altered level of at least one gene expression whencompared to a control, wherein the at least one gene expressed encodesfor a proteoglycan enzyme; (b) correlating the altered level of at leastone gene expression in the sample with the enhanced targeting effect ofcell penetrating peptides; and (c) treating the animal with atherapeutic composition comprising at least one cell penetrating peptideand at least one bioactive agent. In an alternative embodiment, the atleast one gene expressed encodes for an enzyme involved in heparansulfate biosynthesis. Another embodiment provides for the altered levelof at least one gene expression being an elevated level of the at leastone gene expression. Alternatively, the at least one gene having thealtered level of expression is selected from the group consisting ofheparan sulfate 2-O-sulfotransferase 1, exostosin 1, glycosyltransferase8 domain containing 2, heparan sulfate N-deacetylase/N-sulfotransferaseand O-linked N-acetylglucosamine transferase. Alternatively, theenhanced targeting effect is targeted to heparan sulfate. In yet anotherembodiment, the therapeutic binds to heparan sulfate. In still anotherembodiment, the at least one cell penetrating peptide is selected fromthe group consisting of SEQ ID NO:1 and SEQ ID NO:2.

In yet another embodiment, the present invention provides for a methodof treatment, comprising: (a) providing a targeting peptide comprisingat least one amino acid sequence having substantial identity to asequence selected from the group consisting of SEQ ID NO:1 and SEQ IDNO:2; (b) providing at least one bioactive agent which conveys ameasurable therapeutic benefit to the disease; (c) co-administering acomposition comprising (a) and (b) to an animal in need thereof, whereinthe disease is associated with at least one alteration in heparansulfate gene expression levels and the composition comprising (a) and(b) is targeted to at least one heparan sulfate receptor.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “bioactive agent” refers to a substance which is used inconnection with an application that is therapeutic or diagnostic innature, such as in methods for diagnosing the presence or absence of adisease in a patient and/or in methods for treating a disease in apatient. As to compatible bioactive agents, those skilled in the artwill appreciate that any therapeutic or diagnostic agent may beincorporated in the stabilized dispersions of the present invention. Forexample, the bioactive agent may be selected from the group consistingof anti-allergics, bronchodilators, vasodilators, antihypertensiveagents, bronchoconstrictors, pulmonary lung surfactants, analgesics,antibiotics, leukotriene inhibitors or antagonists, anticholinergics,mast cell inhibitors, antihistamines, anti-inflammatories,anti-neoplastics, anesthetics, anti-tuberculars, imaging agents,cardiovascular agents, enzymes, steroids, genetic material, viralvectors, antisense agents, small molecule drugs, proteins, peptides andcombinations thereof. Particularly preferred bioactive agents comprisecompounds which are to be administered systemically (i.e. to thesystemic circulation of a patient) such as small molecule drugs, imagingagents, peptides, proteins or polynucleotides. As will be disclosed inmore detail below, the bioactive agent may be incorporated, blended in,coated on or otherwise associated with the targeting peptide disclosedherein. Particularly preferred bioactive agents for use in accordancewith the invention include anti-allergics, peptides and proteins,bronchodilators, anti-inflammatory agents and anti-cancer compounds foruse in the treatment of disorders involving diseased tissue reflectingaltered heparan sulfate variants specific to said disease. Yet anotherassociated advantage of the present invention is the effective deliveryof bioactive agents administered or combined with a targeting peptide.

The phrase “substantially identical” means that a relevant sequence isat least 70%, 75%, 80%, 85%, 90%, 92%, 95% 96%, 97%, 98%, or 99%identical to a given sequence. By way of example, such sequences may beallelic variants, sequences derived from various species, or they may bederived from the given sequence by truncation, deletion, amino acidsubstitution or addition. Percent identity between two sequences isdetermined by standard alignment algorithms such as ClustalX when thetwo sequences are in best alignment according to the alignmentalgorithm.

A polypeptide “variant” as referred to herein means a polypeptidesubstantially homologous to a native polypeptide, but which has an aminoacid sequence different from that encoded by any of the nucleic acidsequences of the invention because of one or more deletions, insertionsor substitutions. Variants can comprise conservatively substitutedsequences, meaning that a given amino acid residue is replaced by aresidue having similar physiochemical characteristics. See Zubay,Biochemistry, Addison-Wesley Pub. Co., (1983). It is a well-establishedprinciple of protein and peptide chemistry that certain amino acidssubstitutions, entitled “conservative” amino acid substitutions, canfrequently be made in a protein or a peptide without altering either theconformation or the function of the protein or peptide. Such changesinclude substituting any of alanine (A), isoleucine (I), valine (V), andleucine (L) for any other of these amino acids; aspartic acid (D) forglutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) andvice versa; serine (S) for threonine (T) and vice versa; and arginine(R) for lysine (K) and vice versa.

In addition to the known functional variants, there are derivatives ofthe peptides disclosed herein which can also function in the disclosedmethods and compositions. Protein and peptide variants and derivativesare well understood by one of skill in the art and in can involve aminoacid sequence modifications. For example, amino acid sequencemodifications typically fall into one or more of three classes:substitutional, insertional or deletional variants. Insertions includeamino and/or carboxyl terminal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Insertionsordinarily will be smaller insertions than those of amino or carboxylterminal fusions, for example, on the order of one to four residues.Deletions are characterized by the removal of one or more amino acidresidues from the protein or peptide sequence. Typically, no more than 2to 6 residues are deleted at any one site within the protein or peptidemolecule. These variants can be prepared by site-specific mutagenesis ofnucleotides in the DNA encoding the protein or peptide, therebyproducing DNA encoding the variant, and thereafter expressing the DNA inrecombinant cell culture, or via solid state peptide synthesis.

Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known. Amino acid substitutions aretypically of single residues, but can occur at a number of differentlocations at once; insertions usually will be on the order of from 1 to10 amino acid residues; and deletions will range from 1 to 10 residues.Deletions or insertions preferably are made in adjacent pairs, i.e. adeletion of 2 residues or insertion of 2 residues. Substitutions,deletions, insertions or any combination thereof can be combined toarrive at a final construct. The mutations generally should not placethe sequence out of reading frame (unless a truncated peptide isintended) and preferably will not create complementary regions thatcould produce secondary mRNA structure.

Substitutional variants are those in which at least one residue has beenremoved and a different residue inserted in its place. Substantialchanges in function or immunological identity are made by selectingsubstitutions that are less conservative, i.e., selecting residues thatdiffer more significantly in their effect on maintaining (a) thestructure of the polypeptide backbone in the area of the substitution,for example as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site or (c) the bulk of theside chain. The substitutions which in general are expected to producethe greatest changes in the protein properties will be those in which(a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for(or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl,valyl or alanyl; (b) a cysteine or proline is substituted for (or by)any other residue; (c) a residue having an electropositive side chain,e.g., lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine, in this case, (e) byincreasing the number of sites for sulfation and/or glycosylation.Similarly, the term “conformational homology” may be used herein todefine a sequence which maintains a similar arrangement of amino acidsfrom a conformational perspective to SEQ ID NO:1 or SEQ ID NO:2.

As used herein, the term “peptide” is used broadly to mean peptides,proteins, fragments of proteins and the like. The peptide may be animal,bacterial, viral or synthetic in origin. The term “peptidomimetic,” asused herein, means a peptide-like molecule that has the activity of thepeptide upon which it is structurally based. Such peptidomimeticsinclude chemically modified peptides, peptide-like molecules containingnon-naturally occurring amino acids, and peptoids and have an activitysuch as selective homing activity of the peptide upon which thepeptidomimetic is derived (see, for example, Goodman and Ro,Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry andDrug Discovery” Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages803-861). A variety of peptidomimetics are known in the art including,for example, peptide-like molecules which contain a constrained aminoacid, a non-peptide component that mimics peptide secondary structure,or an amide bond isostere. A peptidomimetic that contains a constrained,non-naturally occurring amino acid can include, for example, an.alpha.-methylated amino acid; .alpha.,.alpha.-dialkylglycine or.alpha.-aminocycloalkane carboxylic acid; anN.sup...alpha.-—C.sup..alpha. cyclized amino acid; anN.sup..alpha..-methylated amino acid; a .beta.- or .gamma.-aminocycloalkane carboxylic acid; an .alpha.,.beta.-unsaturated amino acid; a.beta.,.beta.-dimethyl or .beta.-methyl amino acid; a.beta.-substituted-2,3-methano amino acid; an N-—C.sup...epsilon. orC.sup..alpha.-—C.sup..delta. cyclized amino acid; a substituted prolineor another amino acid mimetic. A peptidomimetic which mimics peptidesecondary structure can contain, for example, a non-peptidic .beta.-turnmimic; .gamma.-turn mimic; mimic of .beta.-sheet structure; or mimic ofhelical structure, each of which is well known in the art. Apeptidomimetic also can be a peptide-like molecule which contains, forexample, an amide bond isostere such as a retro-inverso modification;reduced amide bond; methylenethioether or methylene-sulfoxide bond;methylene ether bond; ethylene bond; thioamide bond; trans-olefin orfluoroolefin bond; 1,5-disubstituted tetrazole ring; ketomethylene orfluoroketomethylene bond or another amide isostere. One skilled in theart understands that these and other peptidomimetics are encompassedwithin the meaning of the term “peptidomimetic” as used herein.

As used herein, the term “dendrimer” shall mean repeatedly branched androughly spherical molecules. A dendrimer is typically symmetric around acore and usually adopts a spherical three-dimensional morphology.Dendrimers generally contain three major portions: a core, an innershell and an outer shell. Dendrimers can be synthesized to havedifferent and varying functionality in each of the major portions inorder to control such variables as solubility, thermal stability andattachment of compounds suitable for particular applications.

CARSKNKDC (SEQ ID NO: 1) (CAR) peptide has been previously been shown totarget wound healing (Järvinen and Ruoslahti, 2007). CAR peptide hasalso been linked to decorin for targeted anti-TGF-β scar minimization inskin wounds (Järvinen and Ruoslahti, 2010).

Here we describe the novel homing of CAR peptides to hypertensivepulmonary vasculature, acutely injured pulmonary tissue, and fibroticpulmonary tissue. Additionally, we disclose a novel means of achievingtargeted therapy with CAR via simultaneous administration of CAR peptidealong with another therapeutic.

These findings provide the means to diagnose and deliver targetedtherapies for pulmonary diseases such as pulmonary hypertension,interstitial lung disease, acute lung injury (ALI), acute respiratorydistress syndrome (ARDS), sepsis, septic shock, sarcoidosis of the lung,pulmonary manifestations of connective tissue diseases, includingsystemic lupus erythematosus, rheumatoid arthritis, scleroderma, andpolymyositis, dermatomyositis, bronchiectasis, asbestosis, berylliosis,silicosis, Histiocytosis X, pneumotitis, smoker's lung, bronchiolitisobliterans, the prevention of lung scarring due to tuberculosis andpulmonary fibrosis, other fibrotic diseases such as myocardialinfarction, endomyocardial fibrosis, mediastinal fibrosis,myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis,pneumoconiosis, nephrogenic systemic fibrosis, keloid, arthrofibrosis,adhesive capsulitis, radiation fibrosis, fibrocystic breast condition,liver cirrhosis, hepatitis, liver fibrosis, nonalcoholic fatty liverdisease, nonalcoholic steatohepatitis, sarcoidosis of the lymph nodes,or other organs; inflammatory bowel disease, crohn's disease, ulcerativecolitis, primary biliary cirrhosis, pancreatitis, interstitial cystitis,chronic obstructive pulmonary disease, atherosclerosis, ischemic heartdisease, vasculitis, neoplastic/metastatic/oncological diseases(including cancer), pneumoconiosis, autoimmune diseases, angiogenicdiseases, wound healing, infections, trauma injuries and systemicconnective tissue diseases including systemic lupus erythematosus,rheumatoid arthritis, scleroderma, polymyositis, and dermatomyositis.

These diseases can be treated by simultaneously administering CARpeptide with the bioactive agent to be targeted to the site of disease.We define simultaneous administration, or co-administration, asadministration of CAR followed by administration of the therapeutic tobe targeted within 1 hour of CAR administration. For example, if thedisease is pulmonary hypertension and the desired goal is targetedpulmonary arterial vasodilation, an effective dose of CAR peptide can beco-administered with a minimal dose of systemic vasodilator to achievetargeted pulmonary vasodilation and a significant decrease in pulmonarypressure with minimal systemic hypotension.

Similarly, CAR peptide can be co-administered with other medications toincrease therapeutic bioavailability, boost therapeutic efficacy, andminimize side effects. CAR may be administered in a linear or cyclicalform, or in any conformation deemed physiologically appropriate as ameans of conveying treatment.

In addition to targeted vasodilation, we can also deliver targetedanti-coagulation. For example, in a disease like acute lung injury,which is often marked by pulmonary intra-alveolar coagulation, targetedanti-coagulation can be delivered to the affected pulmonary area byco-administering an effective dose of CAR with an anti-coagulant such astissue factor pathway inhibitor (TFPI) or site-inactivated factor VIIa(Welty-Wolf et al., 2001) in a minimal dose to achieve targetedpulmonary anticoagulation with minimal changes in clotting ability overthe areas of the body not undergoing thrombosis. Selective pulmonaryanti-coagulation can also be utilized to treat other pulmonary diseasesmarked by pulmonary thrombosis such as pulmonary hypertension, lungtransplant rejection and others.

In a disease like chronic obstructive pulmonary disease, which is oftenmarked by shortness of breath, CAR peptide can be co-administered toboost the effective concentration and potency of drugs to relax airwaysmooth muscles such as long lasting 13-2 agonists such as salmeterol orformoterol (Richter, et al., 2002).

Many pulmonary diseases are often marked by a decrease in glutathione(GSH), a powerful antioxidant (Morris and Bernard, 1994). CAR peptidecan be co-administered with N-Acetylcysteine (NAC), a glutathioneprecursor, in diseases like pulmonary fibrosis, PAH, ALI, and otherpulmonary disorders to boost GSH production and scavenge reactiveoxidants often found in pulmonary diseases. GSH may also serve to dampenthe inflammatory immune response by binding to triggering receptorexpressed on myeloid cells 1 (TREM1) and diminishingmonocyte/macrophage- and neutrophil-mediated inflammatory responses.Co-administration of CAR with NAC can serve to lessen the severeinflammatory immune response that often characterizes severe pulmonaryand fibrotic diseases like ALI, pulmonary hypertension, autoimmunediseases and many other conditions.

The levels of antioxidants such as Superoxide Dismutase (SOD)(Rosenfeld, et al., 1996), or synthetic superoxide dismutase mimeticslike EUK-8 (Gonzalez et al., 1996) can be increased throughco-administration of CAR.

Treatments for pulmonary diseases like pulmonary fibrosis, PAH and ALIcan also be improved by co-administering CAR with TGF-β inhibitors likedecorin. Decorin, which has been previously enhanced through directconjugation with CAR (Järvinen and Ruoslahti, 2010), can also beco-administered with CAR to achieve the benefits of targeting withoutdirect conjugation between the CAR and decorin molecules.

In pulmonary hypertension, pulmonary fibrosis and other pulmonarydiseases, the benefits of endothelin (ET-1) receptor antagonists (Kuklinet al., 2004), prostacyclin derivatives (Olschewski et al., 1999),phosphodiesterase type 5 inhibitors (Kanthapillai et al., 2004) andoncological agents such as imatinib (Ghofrani et al., 2005) (Aono etal., 2005) can be increased for patients through the co-administrationof CAR.

Other pulmonary and fibrotic disease treatments such as Ketoconazolewhich inhibits thromboxane and leukotriene synthesis (Sinuff et al.,1999) can be improved in its efficacy while minimizing side effectsthrough co-administration with CAR.

Newer therapeutic approaches such as small interfering RNA (siRNA), andmicroRNA (miRNA) therapies (Wurdinger and Costa, 2007) can also beimproved and side effects minimized through the selective targeting ofdiseased tissue through the co-administration of CAR.

The present invention provides for establishing therapeutic targets byidentifying altered gene expression levels at the heparan sulfatereceptor, therefore indicating a diseased source. The diseased sourcemay then be approached therapeutically with customized targetedtherapies comprising a targeting peptide and bioactive agents disclosedin the present invention.

In addition to targeted therapies, CAR's homing to diseased pulmonaryand fibrotic tissues can be utilized for the purposes of diagnosisthrough the conjugation or co-administration of CAR with imaging agents.

Examples

I. Monocrotaline Pulmonary Hypertension Model:

Animal Model

A rat model of monocrotaline (MCT)-induced pulmonary arterialhypertension was used for this study. Briefly, male Sprague-Dawley rats(150-200 g, Harlan Laboratories, IN) were administered with a singlesubcutaneous injection of monocrotaline at 60 mg/kg (Sigma-Aldrich, MO),while control rats were administered 0.9% saline (FIG. 1). Rats wererandomly selected and studied for peptide distribution studies on 1, 3,7, 14 or 21 days after the treatment of monocrotaline.

Peptides

The following peptides were labeled with 5-carboxyfluorescein (5FAM) andused for the lung targeting studies: CAR, 5FAM-CARSKNKDC; VCAM1,CVHSPNKKCGGSK-5FAM; Control, 5FAM-CGGGGGGGC. All peptides weresynthesized by Anaspec (Anaspec Inc., CA). Peptides were resolved in PBSat the concentrations of 0.5 mg/mL.

Peptide Targeting Study

MCT-treated or untreated rats were injected with peptide solution at adose of 3.3 mg/kg body weight via the tail vein. At two hours after theinjection, rats were perfused with PBS containing 1% bovine serumalbumin under the deep anesthesia with isofluorane at a rate of 3.0% andeuthanized. Tissues were fixed by systemic perfusion with 10% bufferedformalin via right ventricle. The lung was inflated by injection of 10%formalin through the trachea. Various organs were excised from the ratand fixed for additional twenty four hours and processed forimmunohistochemistry.

Immunohistochemistry

To determine the localization of the peptides, paraffin-embedded tissuesections were immunostained with either hematoxylin and eosin (FIGS.2-3) or rabbit anti-fluoroscein isothiocyanate (FITC) antibody(Invitrogen, CA) followed by horseradish peroxidase-labeled anti-rabbitIgG secondary antibody (FIG. 4). The peptide localization was thenvisualized by diaminobenzidine (DAB). An automated staining system,Discovery XT (Ventana, Ariz.) was used. To quantify the targetingefficiency of the peptides to the lung, the immunostained sections werescanned by Aperio Scanscope XT and analyzed using ImageScope software(Aperio Technologies, CA).

II. Targeted Vasodilation in SU5416/Hypoxia/Normoxia-Exposed SevereOcclusive Pulmonary Hypertension:

Animal Model Adult male Sprague-Dawley rats weighing approximately 200 gare injected subcutaneously with SU5416 (20 mg/kg; SUGEN Inc), which issuspended in carboxymethylcellulose (0.5% [wt/vol]carboxymethylcellulose sodium, 0.9% [wt/vol] NaCl, 0.4% [vol/vol]polysorbate, 0.9% [vol/vol] benzyl alcohol in deionized water). The ratsare then exposed to chronic hypoxia in a hypobaric chamber (10% 02) for3 weeks and are returned to normoxia (21% 02) for an additional 2 to 10weeks.

Catheterized Rats

Rats are anesthetized with intramuscular pentobarbital sodium (30mg/kg). The rats are placed on controlled heating pads. Hemodynamicmeasurements are performed in anesthetized animals under normoxicconditions. Polyvinyl catheters (PV-1, internal diameter: 0.28 mm) areinserted into the right jugular vein for measurement of rightventricular systolic pressure (RVSP) and into the left jugular vein fordrug administration. A microtip P-V catheter (SPR-838, MillarInstruments) is inserted into the right carotid artery and advanced intothe left ventrical (LV). The signals are continuously recorded byMPVS-300 system with PowerLab/4SP, A/D converter (AD Instruments), and apersonal computer. RVSP, heart rate, maximal left ventriclar systolicpressure, left ventricular end-diastolic pressure (LVEDP), mean arterialpressure (MAP), cardiac output, and stroke volume are measured. If theheart rate falls below 300 beats/min, the measurements are excluded fromanalysis. At the end of each hemodynamic study, the rat is sacrificed byan overdose of pentobarbital sodium, and organs are removed for variousmeasurements and analyses.

After baseline hemodynamic measurements, a simple mixture of CAR (1mg/300 g rat), or control peptide CARM, and fasudil (0.1, 0.3, 1, or 3mg/kg) or each agent alone is injected intravenously, and allhemodynamic parameters are continuously monitored.

Immunohistochemical Staining

Organs (lung, heart, liver, spleen, and kidney) are collected afterblood is flushed with 30 ml phosphate buffered saline (PBS). Lungs areinflated via trachea with 10% formalin at a constant pressure of 20 cmH₂O. After 24 hour-fixation with 10% formalin, all organs are embeddedin paraffin, and sectioned at 5 mm thickness. After deparaffinization,tissue sections are pretreated with 3% hydrogen peroxidase for 10minutes and blocked with normal horse serum for 1 hour. They are thenincubated for 1 hour with an anti-fluorescein antibody (1:200;Invitrogen) as a primary antibody. After washing with PBS, the sectionswere incubated with biotinylated secondary antibodies, washed with PBS,and incubated in ABC Regent for 5 minutes. Diaminobenzidine was used asa substrate for the immunoperoxidase reaction. Sections were lightlycounterstained with hematoxylin, and analyzed using lightmicroscopically (FIGS. 5-13). CAR (but not CARM) was detected in highintensity in all layers of severely remodeled arteries from lung tissue.Neither CAR nor CARM was found in other organs except for the kidney.

III. Bleomycin-Induced Acute Lung Injury and Pulmonary Fibrosis Model:

The bleomycin (BL) model is usually considered a model of pulmonaryfibrosis, but its administration is also associated with features ofacute lung injury (ALI). Bleomycin is an antineoplastic antibiotic drugisolated in 1966 from the actinomycete Streptomyces verticillus.Bleomycin forms a complex with oxygen and metals such as Fe2+, leadingto the production of oxygen radicals, DNA breaks, and ultimately celldeath. Bleomycin can be inactivated by bleomycin hydrolase, a cysteineprotease that shows variable levels of expression in the lungs. Thesusceptibility of the lungs to bleomycin-induced toxicity is largelydependent on the levels of expression of bleomycin hydrolase in thelungs; species with high levels of expression, such as rabbits, arerelatively resistant to bleomycin-induced toxicity, whereas species withlow levels of expression, such as C57BL/6 mice, are sensitive. Inaddition to species-related differences in bleomycin susceptibility,there are also differences in strain susceptibility, with C57BL/6 micebeing highly sensitive.

Animal Model

A mouse model of bleomycin induced acute lung injury and pulmonaryfibrosis was used for this study. Briefly, 6 WT C57Bl/6 male mice, 8-12weeks were weighed and anesthetized, and given bleomycin (BL)intratracheally at 4 U/kg. At 3 days (acute lung injury model) and 14days (pulmonary fibrosis model) after BL injection, peptides wereinjected via the tail vein.

Peptides

The following peptides were labeled with 5-carboxyfluorescein (5FAM) andused for the lung targeting studies: CAR, 5FAM-CARSKNKDC; VCAM1,CVHSPNKKCGGSK-5FAM; Control, 5FAM-CGGGGGGGC. All peptides weresynthesized by Anaspec (Anaspec Inc., CA). Peptides were resolved in PBSat the concentrations of 0.5 mg/mL.

Peptide Targeting Study

BL-treated mice were injected with peptide solution at a dose of 3.3mg/kg body weight via the tail vein. At two hours after the injection,mice were perfused with PBS containing 1% bovine serum albumin under thedeep anesthesia with isofluorane at a rate of 3.0% and euthanized.Tissues were fixed by systemic perfusion with 10% buffered formalin viaright ventricle. The lung was inflated by injection of 10% formalinthrough the trachea. Various organs were excised from the rat and fixedfor additional twenty four hours and processed for immunohistochemistry.

Immunohistochemistry

To determine the localization of the peptides, paraffin-embedded tissuesections were immunostained with rabbit anti-fluoroscein isothiocyanate(FITC) antibody (Invitrogen, CA) followed by horseradishperoxidase-labeled anti-rabbit IgG secondary antibody. The peptidelocalization was then visualized by diaminobenzidine (DAB). An automatedstaining system, Discovery XT (Ventana, Ariz.) was used. To quantify thetargeting efficiency of the peptides to the lung, the immunostainedsections were scanned by Aperio Scanscope XT and analyzed usingImageScope software (Aperio Technologies, CA) (FIGS. 14-17).

Blood Pressure Tracing

To measure the acute effects of fasudil with and without CARadministration on the right and left ventricular systolic pressure,blood pressure measurements were performed on catheterizedSU5416/hypoxia/normoxia-exposed rats with PAH (FIGS. 18-19).Surprisingly, co-administered CAR enhanced the blood pressure loweringeffect of fasudil on RVSP with only a minor reduction in LVSP, ascompared to fasudil alone. Of additional importance, continuous infusionof CAR+fasudil resulted in a sustained, pulmonary-specific effect evenafter the cessation of the infusion (FIG. 20). An alternative analysiswas conducted, observing the same pulmonary-specific effects whencomparing pressure in the RVSP to systolic aortic pressure (SAP). Whilethe selective decrease in pulmonary pressure as measured in the RVSP ispresent, there is no increased CAR effect systemically whenco-administered with fasudil (FIG. 21).

IV. CAR Variant+Fasudil Analysis:

Animal Model

Severe occlusive PAH rat model was used. Animals were injected withSU5416 (20 mg/kg; SUGEN Inc), followed by 3 weeks hypoxia, then followedby 2-10 weeks normoxia.

Peptides

The peptide administered was a 7 amino acid variant to the CAR peptideused in previous examples. This variant (CARK) consisted of thefollowing sequence: CARSKNK (SEQ ID NO: 2). In these experiments, CARKwas administered at a dose of 3 mg/kg and fasudil administered at 1mg/kg.

Blood Pressure Tracing

To measure the acute effects of fasudil with CARK administration on theright and left ventricular systolic pressure (or systolic aorticpressure), blood pressure measurements were performed on catheterizedSU5w/hypoxia/normoxia-exposed rats with PAH. Similar to CAR, CARKco-administration enhanced the blood pressure lowering effect of fasudilon RVSP with only a minor reduction in SAP (FIG. 22) and LVSP (FIG. 23),as compared to fasudil alone. Interestingly, administration of 10 mg/kgof fasudil 30 minutes after cessation of CARK infusion still resulted ina sustained, pulmonary-specific effect (FIG. 24).

V. CAR+Imatinib Analysis:

Animal Model

Severe occlusive PAH rat model was used. Animals were injected withSU5416 (20 mg/kg; SUGEN Inc), followed by 3 weeks hypoxia, then followedby 2-10 weeks normoxia.

Peptides

The peptide administered was CAR, CARSKNKDC (SEQ ID NO: 1). In thisexperiment, CAR was administered at a dose of 3 mg/kg and imatinibadministered at 10 mg/kg.

Blood Pressure Tracing

To measure the acute effects of imatinib with CAR administration on theright and left ventricular systolic pressure, blood pressuremeasurements were performed on catheterizedSU5w/hypoxia/normoxia-exposed rats with PAH. Similar to fasudil, CARco-administration enhanced the blood pressure lowering effect ofimatinib on RVSP with only a minor reduction in LVSP (FIG. 25).

VI. Altered Levels of Gene Expression of Enzymes Involved in HeparanSulfate Proteoglycan Biosynthesis Found in a Progressive PorcineSurgical Shunt Model of PAH:

Heparan sulfate biosynthetic enzymes are key components in generating amyriad of distinct heparan sulfate structures that carry out multiplebiologic activities. To determine whether CAR or any variants utilizedthe heparan sulfate pathway, an analysis was first performed to identifydifferential gene expression in the PAH model since CAR displayed bothhoming and selective therapeutic efficacy in models of PAH.

It was discovered that in the surgical shunt model of PAH, a largeincrease in gene expression was identified in a select group of genes,all of which are related to the heparan sulfate biosynthetic pathway.The heparan sulfate 2-O-sulfotransferase 1 (HS2ST1) gene, which encodesan enzyme responsible for catalyzing the transfer of sulfate to the C2position of selected hexuronic acid residues within the maturing heparansulfate, was found to be greatly increased over time in the PAH pigmodel (FIG. 26).

Another gene which showed a selective increase in expression in the PAHmodel was exostosin 1 (EXT1), a glycosyltransferase required for thebiosynthesis of heparan sulfate (FIG. 27). Specifically, EXT1 encodes anendoplasmic reticulum-resident type II transmembrane glycosyltranferaseinvolved in the chain elongation step of heparan sulfate biosynthesis.

Other genes identified as exhibiting an increase in expression in thePAH model were glycosyltransferase 8 domain containing 2 (GLT8D2) (FIG.28), heparan sulfate N-deacetylase/N-sulfotransferase (NDST1) (FIG. 29)and O-linked N-acetylglucosamine transferase (OGT) (FIG. 30).

VII. Peptide Variants

It is possible to modify the sequences disclosed in the presentinvention by truncation, i.e., SEQ ID NO:2 is a truncated variant of SEQID NO:1 in which the terminal 2 amino acids of SEQ ID NO:1 are deletedto produce SEQ ID NO:2.

The conformation of peptide variants can be modeled using molecular andelectronic structure modeling programs like MOLDEN.

The molecular and electrostatic potential structure of SEQ ID NO:1 orSEQ ID NO:2 can be modeled, and compared to substitutional variants inwhich one or more amino acids have been substituted to predict if thevariant will have a similar conformation with an expected similarfunction.

SEQ ID NO:2 was modeled in an energy minimized state and anelectrostatic potential map was created to visualize its electrostaticsurface. A library of single amino acid substitutional variants of SEQID NO:2 were modeled by substituting each of the remaining 19 aminoacids for the N terminus cysteine (C) (FIG. 31). The sequences from thelibrary of single amino acid substitutional variants were as follows:AARSKNK (SEQ ID NO:3), NARSKNK (SEQ ID NO:4), SARSKNK (SEQ ID NO:5),TARSKNK (SEQ ID NO:6), MARSKNK (SEQ ID NO:7), GARSKNK (SEQ ID NO:8),VARSKNK (SEQ ID NO:9), LARSKNK (SEQ ID NO:10), IARSKNK (SEQ ID NO:11),HARSKNK (SEQ ID NO:12), FARSKNK (SEQ ID NO:13), WARSKNK (SEQ ID NO:14),YARSKNK (SEQ ID NO:15), QARSKNK (SEQ ID NO:16), EARSKNK (SEQ ID NO:17),DARSKNK (SEQ ID NO:18), RARSKNK (SEQ ID NO:19), KARSKNK (SEQ ID NO:20),PARSKNK (SEQ ID NO:21).

The electrostatic potential conformational structure was comparedbetween SEQ ID NO:2 and each substitutional variant (FIGS. 32-50). Somesubstitutional variants displayed nearly identical or very similarstructures (FIGS. 32-41) indicating a likelihood that substituting theamino acids A, S. M, V, H, T, N, G, L, or I for the N terminus cysteine(C) would result in the substitutional variants AARSKNK (SEQ ID NO:3),SARSKNK (SEQ ID NO:5), MARSKNK (SEQ ID NO:7), VARSKNK (SEQ ID NO:9),HARSKNK (SEQ ID NO:12), TARSKNK (SEQ ID NO:6), NARSKNK (SEQ ID NO:4),GARSKNK (SEQ ID NO:8) and IARSKNK (SEQ ID NO:11) having similarfunctional characteristics as SEQ ID NO:2. Other substitutional variantsshowed greater differences in structure compared to CARK (FIGS. 42-50)with predicted different function.

Other substitutional variants can be modeled and predicted in a similarfashion for SEQ ID NO:1 or SEQ ID NO:2 by substituting any of theremaining 19 amino acids for a particular amino acid in either peptidewith any resulting similar conformers predicted to have similarfunction.

VIII. Dendrimer Variants

Dendrimers are macromolecules having well-defined hyperbranchedstructures.

Peptide dendrimers are radially branched macromolecules that contain apeptidyl branching core and/or peripheral peptide chains, and they canbe divided into three categories. One category consists of “grafted”peptide dendrimers, having peptides only as surface functionalities. Thesecond category is peptide dendrimers composed entirely of amino acids.The third are dendrimers utilizing amino acids in the branching core andsurface functional groups, but having non-peptide branching units.Peptide dendrimers can be synthesized using either divergent orconvergent approach, and the availability of solid-phase combinatorialmethods enables large libraries of peptide dendrimers to be produced andscreened for desired properties.

Dendrimer variants of SEQ ID NO:1 or SEQ ID NO:2 can be synthesized. Forexample, a CARK dendrimer containing 8 CARK residues on a polyamidoamine(PAMAM) core (FIGS. 51-52) can be constructed. Other cores such as Poly(ethylene glycol) can also be used to create dendrimer variants. Thesedendrimer variants can have dozens, hundreds, or even thousands of CARor CARK peptide residues on the surface of the dendrimer to provideenhanced functional characteristics. These dendrimers can contain CAR orCARK alone for disease selective homing, cell penetration and deliveryof co-administered bioactive agents or contain a bioactive agent withinthe dendrimer for targeted delivery.

REFERENCES

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What is claimed is:
 1. A composition comprising: (a) a targeting peptideconsisting of the amino acid sequence of SEQ ID NO:2; and (b) at leastone bioactive agent which conveys a therapeutic benefit to a disease,wherein the at least one bioactive agent is selected from the groupconsisting of antiallergics, bronchodilators, antifibrotics,antihypertensive agents, bronchoconstrictors, pulmonary lungsurfactants, analgesics, antibiotics, leukotriene inhibitors orantagonists, anticholinergics, mast cell inhibitors, anti-neoplastics,prostacyclins, anesthetics, anti-tuberculars, imaging agents,cardiovascular agents, enzymes, steroids, viral vectors, antisenseagents, small molecule drugs, proteins, and peptides, and wherein thetargeting peptide and the at least one bioactive agent are bound.
 2. Thecomposition of claim 1, wherein the disease is selected from the groupconsisting of pulmonary hypertension, acute respiratory distresssyndrome (ARDS), sepsis, septic shock, sarcoidosis of the lung, systemiclupus erythematosus, rheumatoid arthritis, scleroderma, polymyositis,dermatomyositis, bronchiectasis, asbestosis, berylliosis, silicosis,Histiocytosis X, pneumotitis, bronchiolitis obliterans, myocardialinfarction, endomyocardial fibrosis, mediastinal fibrosis,myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis,nephrogenic systemic fibrosis, keloid, arthrofibrosis, adhesivecapsulitis, radiation fibrosis, fibrocystic breast condition, livercirrhosis, hepatitis, nonalcoholic fatty liver disease, nonalcoholicsteatohepatitis, sarcoidosis of the lymph nodes, Crohn's disease,ulcerative colitis, primary biliary cirrhosis, pancreatitis,interstitial cystitis, chronic obstructive pulmonary disease, and woundhealing.
 3. The composition of claim 1, wherein the at least onebioactive agent is specific for the disease being treated.